Autosomal dominant polycystic kidney disease (ADPKD) is largely caused by mutations in the PKD1 and PKD2 genes, encoding the polycystins 1 (PC1) and 2 (PC2) proteins. The pathogenesis of ADPKD entails dysfunctional ion transport and/or regulatory mechanisms that impair ion transport, in particular calcium, mediated by PC2 in renal epithelia and other target tissues. PC2 is a member of the TRP superfamily of ion channels, and behaves as a calcium-permeable, non-selective cation channel implicated in signaling events that lead to calcium-influx induced cell activation. We previously described the functional properties of the PC2 channel, including its multiple conductance substates, voltage dependence, and regulation by the C-terminal tail of PC1. PC2 permeates calcium, which also inhibits its channel activity, and is inhibited by hydrogen ions. PC2 also interacts and forms functional complexes with other members of the TRP family of channels, providing functional diversity in cell signaling. Thus, PC2 channel function and regulation are central to a number of signaling mechanisms that regulate cell function and growth in renal epithelial cells and possibly other cells. How PC2 permeates and regulates calcium transport (and other ions) remains largely unknown, as are also largely unknown the subunit composition of the channel and regulatory mechanisms of its channel function. Here, we propose to further investigate the biophysical properties of the PC2 ion channel as a means to better understand how PC2 transports, and elicits calcium signals. We plan to use a number of technologies including new methods of isolating and purifying PC2, in combination with electrophysiological, molecular biological and biochemical methodologies and atomic force microscopy to determine the structure/function correlates of the PC2 channel complex. In this revised application we obtained the preliminary information that makes these studies novel and unique, allowing for the simultaneous imaging of functioning PC2 channels, both alone, and in combination with other members of the TRP family. Here, we propose to determine how PC2 monomers assemble into a functional channel complex, and how PC2 interacts, and modifies its functional properties by interacting with other TRP channels. Further we will determine how these interactions modify both the topological features of the channel, as well as its biophysical properties, including cation selectivity, gating, substate residence times, voltage dependence and sensitivity to blockers. These studies will provide a new and more complete understanding of PC2 function, as well as define the molecular basis for functional diversity, which makes its contribution relevant to cyst formation and expansion that characterize a number of cystic diseases, including ADPKD. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD) describes genetic disorders, collectively affecting 1:1,000 of the world's population. ADPKD patients suffer from hypertension and end-stage renal disease. ADPKD is caused by mutations in the PKD1 and PKD2 genes encoding polycystins 1 and 2. Our proposed studies will determine how polycystin-2 functions as a channel, and how it interacts with other proteins. Our studies will provide a more complete understanding of polycystin-2 function, and will define the molecular basis of cystic diseases, including ADPKD.